The complete pathway for microbial denitrification has been established as: EQU NO.sub.3.sup.- .fwdarw.NO.sub.2.sup.- .fwdarw.NO.fwdarw.N.sub.2 O.fwdarw.N.sub.2
(Ye et al., Appl. Environ. Microbiol. 60:1053-1058(1994); Zumft et al., Microbiol. Mol. Biol. Rev. 61:533-616 (1997)). In the natural environment, denitrification plays a major role in completing the nitrogen cycle by converting nitrate (NO.sub.3.sup.-) or nitrite (NO.sub.2.sup.-) to nitrogen gas (N.sub.2). Bacteria attempt to maintain the balance necessary between fixed nitrogen and atmospheric nitrogen. In the denitrification process, the soil bacteria use nitrate, rather than oxygen, as the ultimate electron acceptor in a series of reactions to generate a transmembrane proton gradient that is used to synthesize ATP.
In practical applications, microbial denitrification has been widely used for water purification (Mateju et al., Enzyme Microb. Technol. 14:172-183 (1992)). On the other hand, nitrous oxide (N.sub.2 O) has been shown to have detrimental effect on the stratospheric ozone layer (de Boer et al., Eur. J. Biochem. 242:592-600 (1996)). NOx, along with carbon monoxide and hydrocarbons can lead to an increase in the amount of stratospheric ozone. Thus, the production of N.sub.2 O and nitric oxide (NO) due to incomplete denitrification is of concern. It will be useful therefore to devise new and better methods for denitrification of industrial waste streams to effect complete denitrification. The identification of genes encoding proteins responsible for key denitrification reactions will be essential for the development of improved denitrification methods.
Two of the essential genes in the bacterial denitrification pathway are those encoding nitric oxide reductase (nor) and nitrate reductase (nap). Genes encoding these enzymes have been identified in both denitrifying bacteria as well as non-denitrifyers. For example, Bartnikas et al., J. Bacteriol. 179:3534-3540 (1997) teach the identification and sequencing of a gene cluster required for the expression of nitric oxide reductase in Rhodobacter sphaeroidesi and de Boer et al., (Eur. J. Biochem. 242:592-600 (1996)) describe a nor gene cluster isolated from Paracoccus denitrificans. Genes encoding periplasmic nitrate reductase have been characterized from Thiosphaera pantotropha (Berks et al., Biochem J., (1995), 309, 983) and from Rhodobacter sphaeroides (Reyes et al., Biochem J, (1998), 331, 897). Finally Grove et al., (Mol. Microbiol. 19:467-481 (1996)) describe the identification of a gene encoding a periplasmic nitrate reductase from the non-denitrifying E. coli K-12.
The utility in being able to manipulate the nor and nap genes to modify bacterial denitrification is clear from the interplay of these enzymes with the genes encoding nitrite reductase (nir) in the bacterial denitrification process. In bacterial denitrification, NO is produced from NO.sub.3.sup.- in two consecutive reactions catalyzed by the two metalloenzymes nitrate reductase and nitrite reductase, and then is decomposed into N.sub.2 O by nitric oxide reductase. The key step of denitrification is the reduction of NO.sub.2.sup.- by nitrite reductases. These quintessential enzymes catalyze the conversion of a mineral form of nitrogen to a gaseous form. It is well recognized that gaseous forms of nitrogen compounds are no longer easily available for assimilation by the biomass.
The product of nitrite reduction, NO, is only present in trace amount due to its efficient removal by nitric oxide reductase. However, it has been observed that mutations in the NO step render the cells incapable of using nitrate and nitrite as the alternative electron acceptors due to NO toxicity. In addition, mutations in the nitric oxide reductase (nor) region have shown a negative impact on the activity or expression level of nitrite reductase (nir) in Pseudomonas stutzeri and Paracoccus denitrificans (Grove et al., Mol. Microbiol. 19:467-481 (1996); Zumft et al., Eur. J. Biochem. 219:481-490 (1994)). Nonetheless, in Rhodobacter sphaeroides, promoter activity for the nitrite reductase gene nirK was higher in nor mutants (Bartnikas et al., J. Bacteriol. 179:3534-3540 (1997)). Activities of these two steps are commonly controlled by the proteins NnrU, NirQ, and NnrR (Bell et al., FEBS Lett. 265:85-87 (1990); Kastrau et al., Eur. J. Biochem. 222:293-303 (1994); Tyson et al., Arch. Microbiol. 168:403-411 (1997)), depending on the organism studied. This evidence distinctly suggests that reduction of nitrite and nitric oxide is highly interdependent.
Prior to the identification of role of the nitrate reductases in the periplasmic space, the principal nitrate reductase activity in bacterial denitrification has been attributed to those associated with membrane (Bell et al, 1993, J. of General Microbiology, Vol. 139, p. 3205-3214).
Applicant has discovered new genes isolated from Pseudomonas sp. encoding a periplasmic nitrate reductase and nitric oxide reductase. The genes are may be expressed in recombinant systems effect bacterial denitrification and the genes or portions thereof may be used to identify other denitrifying bacterial strains. Further, it has been demonstrate that the instant periplasmic nitrate reductase is the penultimate nitrate reductase in the denitrification pathway as compared with the cytoplasmic variety of the enzyme in Pseudomonas sp.